This proposal involves three, presumably homologous, low molecular weight proteinase inhibitors, one of which inhibits pancreatic carboxypeptidase, the second chymotrypsin, and third exhibits primary specificity toward trypsin. Attempts will be made to prepare crystals of all three inhibitors to be used in X-ray diffraction studies. The amino acid sequence, disulfide bond pairs, and reactive site of the trypsin inhibitor and the reactive site and properties of the nitro derivative of the chymotrypsin inhibitor will be determined to provide data similar to those already available for the carboxypeptidase inhibitor. In addition, the extremely tight association between proteinase inhibitors and target enzymes will be utilized to prepare these enzymes by affinity chromatography on immobilized inhibitors. Because of the paucity of effective affinity systems for the carboxypeptidases, emphasis will be placed on the identification and purification of suitable members of this enzyme class. Finally, affinity label derivatives of protease inhibitors will be synthesized to provide reagents which are highly specific for their target enzymes and which are irreversible in their interactions.